Thermo Fisher Scientific 293 SFM II User guide

Brand: Thermo Fisher Scientific

Model: 293 SFM II

Type: User guide

Publication number MAN0007369 Rev. 2.00

293 SFM II (1X)

Description

293 SFM II is a serum-free, human, and animal-origin free, low-protein (<10 µg/mL) formulation optimized for high-density suspension

culture of 293 (human embryonic kidney) cells. 293 SFM II supports HEK 293 cellular production of adenovirus and/or glycosylated

recombinant protein, comparable to levels produced in serum-supplemented media. 293 SFM II is not recommended for adherent 293 cell

cultures. 293 SFM II has been demonstrated to support the growth of Per.C6® cells and the high-density suspension culture of HeLa S3 cells.

Product Catalog no. Amount Storage Shelf life*

293 SFM II (1X), liquid 11686-029 1000 mL 2°C to 8°C; Protect from light 6 months

* Shelf life duration is determined from Date of Manufacture.

Product use

For Research Use Only. Not for use in diagnostic procedures.

Important information

293 SFM II facilitates the adaptation of monolayer dependent

293 cells to growth in suspension culture.

Safety information

Read the Safety Data Sheets (SDSs) and follow the handling

instructions. Wear appropriate protective eyewear, clothing, and

gloves.

Prepare medium

• 293 SFM II Medium requires aseptic supplementation with

4 mM L-glutamine or GlutaMAX™-I prior to use.

• Antibiotics are not recommended; however, 5 mL/L of

Penicillin-Streptomycin (100X) may be used when required.

• Addition of a surfactant such as Pluronic® F-68 is not required.

Culture conditions

Media: Complete 293 SFM II Medium.

Cell line: HEK 293 cells.

Culture type: Suspension

Culture vessels: Shake flask, spinner bottle, or bioreactor.

Temperature range: 36°C to 38°C.

Incubator atmosphere: Humidified atmosphere of 8% CO2 in air.

Ensure proper gas exchange and minimize exposure of cultures to

light.

Adapt to suspension culture in 293 SFM II

Note: We offer 293-F and 293-H cells that have been pre-adapted

to growth in 293 SFM II.

1. Aspirate media from cell monolayer and displace 293 cells

from the flask surface by rapping the flask sharply against

your hand or a protected surface several times.

Note: Do not use trypsin or other proteolytic agents to

dislodge cells.

2. Resuspend dislodged cells in 5 mL of 293 SFM II.

Note: 293 cells cultured in 293 SFM II may grow as clusters of

2–10 cells.

3. Disperse clusters into a single-cell suspension by triturating

with a small bore pipette or vortexing before passaging or

counting. Optimal vortexing conditions must be determined

based upon speed and duration versus viability.

4. Determine viable cell density using a Countess® Automated

Cell Counter. Alternate methods (e.g., Coulter counter or

hemocytometer) may also be used.

5. Dilute cells in prewarmed complete 293 SFM II to a viable cell

density of 1 × 106 cells/mL. Dispense cell suspension, up to a

maximum of 30 mL/flask, into sterile 125-mL Erlenmeyer

shake flasks.

6. Incubate the shake flask(s) on a rotary shaker (125–130 rpm)

at 37°C in a humidified atmosphere of 8% CO2 in air.

Note: Cells will not thrive in 293 SFM II at lower CO2 levels

(e.g., 5% CO2).

7. When the viable cell density reaches ~1.5 × 106 cells/mL, dilute

the cells with prewarmed, complete 293 SFM II to 2.5 × 105–

3.0 × 105 cells/mL.

8. After the first passage, dilute cells to 2.5 × 105–3.0 × 105

cells/mL whenever the viable cell density exceeds0.75 × 106–

1 × 106 cells/mL. After several passages of consistent growth

and viability in 293 SFM II, the culture is considered to be

adapted.

Note: After adaptation to growth in serum-free suspension

culture, it is possible to scale-up the cultures in spinner flasks or

bioreactors. The appropriate spinner or impeller speed should be

individually determined.

Caution: Some spinner apparatus emit significant heat and

water-jacketed incubators usually cannot readily equilibrate to

temperature variations. Temperatures >40°C are lethal to

HEK 293 cells.

Transfection protocol

Important: Complex formation of DNA with transfection

reagents such as Lipofectamine® Plus and Lipofectamine® 2000

are inhibited by constituents of 293 SFM II. Therefore, these

transfection reagents and media should not be used together.

Prepare DNA-liposome complexes:

1. To each well of a six-well plate, add 1 mL Opti-MEM®

Reduced Serum Medium.

2. For each transfection, add 3–4 µg of DNA to each well.

Gently swirl the plate to mix.

3. For each transfection, add 10–20 µL of Lipofectamine® 2000

transfection reagent to each well. Gently swirl to mix the

DNA and lipid.

4. Incubate at room temperature for 20–45 minutes, and

repeatedly gently swirl the plate allowing DNA-liposome

complexes to form. Although the solution may appear

somewhat cloudy it will not impair transfection.

For additional technical information such as Safety Data Sheets (SDS), Certificates of Analysis, visit www.lifetechnologies.com/support.

For further assistance, email techsupport@lifetech.com.

specified. Per.C6 is a trademark of Crucell Holland B.V.

DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED,

INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW,

IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER

BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING

BUT NOT LIMITED TO THE USE THEREOF.

www.lifetechnologies.com

Prepare 293 cells for transfection:

1. Determine the viable cell density of a suspension culture of

293 cells growing in 293 SFM II.

Note: Cells should be in mid-log phase growth prior to

transfection for optimal results.

2. Calculate the volume of cell suspension required to plate

293 cells into six-well plates at 2 × 106 cells/well. Include two

wells (4 × 106 cells) overage. Transfer this volume of cell

suspension to a sterile 50-mL centrifuge tube. Bring the total

volume to 50 mL with Opti-MEM® Reduced Serum Medium.

3. Centrifuge cell suspension at 200 × g for 5 minutes. Aspirate

and discard the supernatant, being sure not to disturb the cell

pellet.

4. Resuspend the cell pellet in 10 mL Opti-MEM® Reduced

Serum Medium, pipetting up and down to achieve a single-

cell suspension. Add Opti-MEM® Reduced Serum Medium to

a total volume of 50 mL.

5. Centrifuge cell suspension at 200 × g for 5 minutes. Aspirate

and discard the supernatant, being sure not to disturb the cell

pellet.

Important: This second wash step is imperative for optimal

transfection efficiency.

6. Resuspend the cell pellet in 200 µL of Opti-MEM® Reduced

Serum Medium for every 2 × 106 cells contained in the tube.

This will yield a suspension of 1 × 107 cells/mL.

7. Add 200 µL of cell suspension to each well of the six-well plate

containing DNA-liposome complexes. Pipet up and down to

mix well.

8. Incubate at 37°C for 5 hours in a humidified 8% CO2 incubator.

There is no need to remove the transfection mixture, or to feed

with growth medium.

Note: Transfection procedure can be scaled up or down by

adjusting the amount of DNA, Lipofectamine® 2000 transfection

reagent and cell concentration in proportion to the difference in

surface area of the culture vessel.

Transient expression

Harvest and assay cell extracts or stain cells in situ for reporter gene

activity at 24 hours after the start of transfection.

Stable expression

1. Passage the cells at 24 hours post-transfection using the same

seeding density or split ratio that is normally used.

2. At 48 hours post-transfection, replace spent medium with

medium containing the appropriate selective antibiotic

(e.g., Geneticin®).

Note: Suspension cells will need to be collected by

centrifugation (100 × g for 4 minutes) before changing spent

medium for medium containing selective antibiotic.

Thermo Fisher Scientific 293 SFM II User guide

Brand: Thermo Fisher Scientific

Model: 293 SFM II

Type: User guide

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Thermo Fisher Scientific 293 SFM II User guide

Thermo Fisher Scientific 293 SFM II User guide

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